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  Apparatus, methods and compositions for biotechnical separationsUSPTO Application #: 20060141521Title: Apparatus, methods and compositions for biotechnical separations Abstract: RNA, preferably messenger RNA, is purified by use of selective precipitation, preferably by addition of compaction agents. Also included is a scaleable method for the liquid-phase separation of DNA from RNA. RNA may also be recovered by fractional precipitation according to the invention. We have discovered that specific classes of compounds are unexpectedly potent in causing selective precipitation of DNA away from RNA, at low concentrations and in the presence of relatively elevated ionic strength. Modification of the selective removal of DNA can also remove both RNA and DNA, leaving behind a mixture which is advantageous for the further purification of, e.g., proteins. Additional aspects of the invention include mini-preps, preferably of RNA or of plasmid and chromosomal DNA to obtain sequenceable and restriction digestible DNA in high yields in multiple simultaneous procedures. Still further aspects disclose enhanced stripping of the compaction agent by a stripping method comprising high salt addition and pH shift, and combinations of these techniques. RNA Abstract Additionally, a new approach to the isolation of RNA from bacterial lysates employs selective precipitation by compaction agents. The above techniques can be enhanced by use of phase transfer catalysts (PTCs), most preferably selected polyamines of U.S. Pat. No. 6,617,108 polyamines which are quaternary compounds. (The use of PTCs in biotechnical processes is not evident in the literature, see e.g. www./ptorganics.com as of 27 Jan. 2005.) (end of abstract) Agent: Richard Coale Willson Jr - Palm Harbor, FL, US Inventors: Richard C. Willson, Richard Don Goodin USPTO Applicaton #: 20060141521 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060141521. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The present application claims priority of U.S. provisional patent application 60/849,896 filed Feb. 3, 2005 and is a continuation-in-part of U.S. patent application Ser. No. 09/841,763 filed Apr. 24, 2001 which is itself a continuation-in-part of 09/609,996 filed Jul. 3, 2000, now U.S. Pat. No. 6,617,108 granted 9 Sep. 2003, which itself has priority of U.S. Provisional Application 60/143,768 filed Jul. 12, 1999. BACKGROUND OF THE INVENTION [0002] I. Field of the Invention [0003] The present invention relates to the general field of biochemical assays and separations, and to apparatus for their practice, generally classified in U.S. Patent Class 435. [0004] II. Description of the Prior Art [0005] Interest in nucleic acid purification and assays has increased with human trials of plasmid-based vaccines (e.g., for influenza, HIV, and malaria) and therapeutics (e.g., insulin and vascularization promoters) as well as the steady expansion of DNA sequencing activities, pharmacogenomics, and genetic testing. This invention embodies a rapid and efficient method of nucleic acid assay using selective precipitation by compaction agents. [0006] Prior Art will include the following: [0007] Murphy, J. C., Wibbenmeyer, J. A., Fox, G. E., and Willson, R. C. (1999) Purification of plasmid DNA using selective precipitation by compaction agents. Nature Biotechnol. 17, 822-823. [0008] Murphy, J. C., G. E. Fox and R. C. Willson, "RNA Isolation and Fractionation using Compaction Agents," Analytical Biochemistry, 295, 143 (2001). [0009] Mourich D. V., Munks M. W., Murphy J. C., Willson R. C., and Hill A. B., "Spermine compaction is an efficient and economical method of producing vaccination-grade DNA", J. Immunol. Methods. 274:257-264 (2003). [0010] DeWalt, B., Murphy, J. C., Fox, G. E., and Willson, R. C., "Compaction Agent Clarification of Microbial Lysates", Protein Expression and Purification, 28(2) 220-223 (2003). [0011] Related U.S. Pat. No. 6,617,108 describes and claims processes for purification of DNA and separation of proteins, etc. by adding a compaction agent e.g. basic polypeptides, polyamines, trivalent and tetravalent metal ions to the mixture. This present application embodies the discovery that unexpectedly superior results are obtained by use of selected compaction agents e.g. quaternary ammonium compounds and the phosphonium-based catalysts. Preferred compaction agents include quaternary ammonium polyamines, the compounds of FIGS. 1-3. Tables B, C and D show some examples of these compounds. [0012] In the literature, "q-value" and "C#" are useful parameters for correlating reactivity and selectivity. The q-value was proposed by Marc Halpern (PTC Organics) in 1988 and reflects the accessibility of the positive charge of a quat. A q-value of 1.0 to 2.0 often gives high reactivity for "transfer rate limited" reactions whereas a q-value of <1 often gives high reactivity for reactions which require a loose ion pair. The q-value is calculated by adding the reciprocals of the numbers of carbons on each of the 4 chains. C# is simply the sum of all of the carbons of the quat and is a measure of the organophilicity of the quat. Sources include Sachem, Cognis, Lion Akzo, Merck and PTC organics, whose websites give additional information. [0013] III. Problems Presented by Prior Art [0014] Most current methods of nucleic acid separation are relatively time-consuming and require the use of adsorbents, toxic substances, nucleases, and/or filtration media to separate individual nucleic acid types from protein, genomic DNA, endotoxins and especially the abundant RNA present in cell lysates. Many do not provide samples in a state ready for assays such as RT-PCR. [0015] The present invention offers several important improvements over current methods: no DNAse and/or other enzymes need be used, the technique requires no chromatographic medium, and the purified samples are left in a form ready for assays such as RT-PCR. Also, with the use of different compaction agents, different types of nucleic acids can be separated from the same mixture. The invention can separate different types of RNA and DNA as long as some secondary structure is present. [0016] In addition, RNA can be fractionated based on molecular weight via selective precipitation. [0017] The method can also be used for parallel purification of a large number of samples (mini-preps) and is readily adaptable to automation (robotics). [0018] In a preferred embodiment, the invention also provides a method for making a biochemical assay of one or more selected messenger RNA (mRNA) species, while reducing the RT-PCR background due to genomic DNA contamination. [0019] The invention also provides a method of preparing a nucleic acid sample having reduced DNA content and also reduced content of compaction precipitation agent by selective removal of compaction agent. [0020] Especially preferred embodiments include selective DNA precipitation over RNA, assay of RNA by first precipitating DNA, then assaying RNA in a second step; syntheses of preferred compaction precipitation agents; and removal of compaction agents before performing biochemical assays on the sample. SUMMARY OF THE INVENTION [0021] The invention comprises a method of assaying RNA in a mixture comprising DNA and RNA, said method comprising in combination the steps of: A. adding a compaction agent to effect compaction precipitation; and B. adding an enzyme to catalyze a reaction; and C. detecting a product of the reaction catalyzed by the enzyme. [0022] The invention further comprises a method of preparing substantially purified RNA without the use of nucleases or proteases, or organic solvent extraction, comprising adding an effective amount of a compaction agent selected from the group consisting of: quaternary ammonium polyamines and Phase Transfer Catalysts (PTCs) to a lysate containing DNA and RNA to selectively precipitate from said lysate, plasmid-DNA, chromosomal-DNA, or oligonucleocidal-DNA having a content of RNA of less than 3% by weight. [0023] Compositions of the invention comprise a mixture comprising RNA, DNA, a compaction precipitation agent, and a reverse transcriptase or DNA polymerase enzyme. Continue reading... Full patent description for Apparatus, methods and compositions for biotechnical separations Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Apparatus, methods and compositions for biotechnical separations patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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